Osteoporosis is a major public health problem, and it is especially prevalent in aging populations (1, 24, 31). The majority of fractures that occur in people over the age of 65 are due to osteoporosis (24, 60). Peak bone mass is a determining factor in establishing the risk of osteoporotic fracture (Heaney et al., 2000), and studies indicate that genetic factors contribute significantly to the variance in peak bone mass. One of the genes that regulate bone mass has recently been identified via positional cloning. Loss of function mutations in low density lipoprotein receptor-related protein 5 (LRP5), a co-receptor for the canonical Wnt signaling pathway (39), were found to be associated with Osteoporosis-Pseudoglioma Syndrome (OPPG), an autosomal recessive disorder which shows a reduction of bone density in humans (14). In addition, two independent kindreds that manifest familial High Bone Mass (HBM) phenotypes were found to harbor a Gly171 to Val substitution mutation (G171V) in LRP5 (5, 32). More recently, additional HBM mutations were reported in the same structural domain of the G171V mutation (51). Moreover, mice in which the LRP5 genes were inactivated by gene targeting showed phenotypes similar to those of OPPG patients (25), and transgenic expression of LRP5G171V in mice resulted in HBM (2). Furthermore, mouse primary osteoblasts showed reduced responsiveness to Wnt in the absence of LRP5 (25), and Wnt (14) or activated β-catenin (4) stimulated the canonical Wnt signaling activity and induced the production of the osteoblast marker alkaline phosphatase (AP) in osteoblast-like cells. Together, these pieces of evidence indicate that the canonical Wnt signaling pathway plays an important role in the regulation of bone development.
Until recently, the canonical Wnt signaling pathway was believed to start when Wnt bound to frizzled Fz proteins. The seven transmembrane domain-containing Fz proteins suppress the Glycogen synthase kinase 3 (GSK3)-dependent phosphorylation of β-catenin through ill-defined mechanisms involving Dishevelled proteins. This suppression leads to the stabilization of β-catenin. β-catenin can then interact with transcription regulators, including lymphoid enhancing factor-1 (LEF-1) and T cell factors (TCF), to activate gene transcription (10, 15, 56). Recently, genetic and biochemical studies have provided solid evidence to indicate that co-receptors are required for canonical Wnt signaling in addition to Fz proteins (39, 40). The fly ortholog of LRP5/6 (LRP5 or LRP6), Arrow, was found to be required for the signaling of Wg, the fly ortholog of Wnt-1 (54). LRP5 and LRP6 are close homologues which basically function the same way, yet exhibit, different expression patterns. In addition, LRP6 was found to bind to Wnt 1 and regulate Wnt-induced developmental processes in Xenopus embryos (48). Moreover, mice lacking LRP6 exhibited developmental defects that are similar to those caused by deficiencies in various Wnt proteins (42). Furthermore, LRP5, LRP6 and Arrow were found to be involved in transducing the canonical Wnt signals by binding Axin and leading to Axin degradation and β-catenin stabilization (30, 50). The LRP5/6-mediated signaling process does not appear to depend on Dishevelled proteins (28, 45). Recently, a chaperon protein, Mesd, was identified as required for LRP5/6 transport to the cell surface (9, 19).
Xenopus Dickkopf (Dkk)-1 was initially discovered as a Wnt antagonist that plays an important role in head formation (13). Thus far, four members of Dkk have been identified in mammals (26, 37). These include Dkk1, Dkk2, Dkk3 and Dkk4. Dkk1 and Dkk2 inhibit canonical Wnt signaling by simultaneously binding to LRP5 or LRP6 and a single transmembrane protein Kremen (3, 34, 35, 46). It has been previously reported that the LRP5 HBM G171V mutation appeared to attenuate Dick 1-mediated antagonism to the canonical Wnt signaling (5). The present invention describes the mechanism for this attenuation.